Modified hepatitis post-transcriptional regulatory elements

Abstract

Provided are polynucleotides containing a modified PRE having a variant X gene that includes one or more stop codons not present in an unmodified, such as wild-type, hepatitis X gene. Also provided are polynucleotides containing a modified PRE having a variant X gene that includes one or more degradation sequences not present in an unmodified, such as wild-type, hepatitis X gene. The modified PRE can be operably linked to a nucleic acid encoding a recombinant protein. Also provided are expression cassettes, viral vectors and cells containing the polynucleotides, and compositions and methods of use thereof.

The invention claimed is: 
     
       1. A polynucleotide, comprising a modified post-transcriptional regulatory element (PRE), said modified PRE comprising a variant of a wild-type hepatitis virus X gene, said variant X gene comprising a plurality of stop codons not present in the wild-type X gene, wherein the variant X gene comprises a stop codon in each reading frame present in said variant X gene. 
     
     
       2. The polynucleotide of  claim 1 , further comprising a nucleic acid encoding a recombinant protein operably linked to the modified PRE. 
     
     
       3. A vector comprising the polynucleotide of  claim 2 . 
     
     
       4. A cell comprising the polynucleotide of  claim 2 . 
     
     
       5. A polynucleotide, comprising a modified post-transcriptional regulatory element (PRE), said modified PRE comprising a variant of a wild-type hepatitis virus X gene, said variant X gene comprising a plurality of stop codons not present in the wild-type X gene; and,
 a nucleic acid encoding a recombinant protein that is a recombinant receptor operably linked to the modified PRE. 
 
     
     
       6. The polynucleotide of  claim 5 , wherein the stop codon comprises at least one stop codon selected from among:
 a stop codon beginning at a position within or within at least 9, 12, 15, or 18 nucleotides in the 3′ direction from a position in the variant X gene corresponding to the 5′ position of a start codon of the X protein open reading frame; and/or 
 a stop codon beginning at a position within or within at least 9, 12, 15, or 18 nucleotides in the 3′ direction from a position in the variant X gene corresponding to residue 411 of WHV post-transcriptional regulatory element (WPRE) sequence set forth in SEQ ID NO: 1 and/or residue 1503 of the WHV sequence set forth as SEQ ID NO: 2. 
 
     
     
       7. The polynucleotide of  claim 5 , wherein the variant X gene further comprises a sequence encoding a post-translational modification signal not present in the wild-type hepatitis virus X gene. 
     
     
       8. The polynucleotide of  claim 5 , wherein said variant X gene is a variant of a wild-type mammalian hepatitis virus X gene. 
     
     
       9. The polynucleotide of  claim 8 , wherein said wild-type mammalian hepatitis virus X gene is a wild-type woodchuck hepatitis virus (WHV) X gene. 
     
     
       10. The polynucleotide of  claim 5 , wherein the modified PRE comprises nucleotide modifications compared to a wild-type or unmodified hepatitis virus PRE that is a mammalian hepatitis PRE. 
     
     
       11. The polynucleotide of  claim 10 , wherein said wild-type or unmodified mammalian hepatitis PRE is a wild-type woodchuck hepatitis virus PRE (WPRE). 
     
     
       12. The polynucleotide of  claim 10 , wherein the wild-type or unmodified hepatitis virus PRE comprises:
 a) the sequence of nucleotides set forth in SEQ ID NO:1 or 125 or a sequence of nucleotides that exhibits at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1 or 125; or 
 b) a portion of the sequence of nucleotides of a), wherein the portion exhibits post-transcriptional activity. 
 
     
     
       13. The polynucleotide of  claim 5 , wherein the modified PRE is selected from among:
 a) a modified PRE comprising a sequence of nucleotides that exhibits at least 95% sequence identity to SEQ ID NO:1 or 125, said modified PRE containing a variant X gene comprising at least one stop codon not present in SEQ ID NOS:1 or 125; and 
 b) a modified PRE comprising a portion of the sequence of nucleotides of a), said portion comprising a variant X gene comprising the plurality of stop codons, wherein the portion exhibits post-transcriptional activity. 
 
     
     
       14. The polynucleotide of  claim 5 , wherein the variant X gene comprises the sequence of nucleotides set forth in any of SEQ ID NOS: 44-54 or 141-151 and/or the modified PRE comprises the sequence of nucleotides set forth in any of SEQ ID NOS:29-39 or 126-136. 
     
     
       15. The polynucleotide of  claim 5 , wherein said variant X gene further comprises a variant of a start codon comprising one or more nucleotide differences compared to a wild-type hepatitis virus X gene start codon and/or compared to the start codon corresponding to nucleotide positions 411-413 of SEQ ID NO: 1, wherein the one or more nucleotide differences results in restricted or prevented translation initiation from said start codon. 
     
     
       16. The polynucleotide of  claim 5 , wherein said variant X gene comprises a variant promoter operably linked to said variant X gene, said variant promoter comprising one or more nucleotide differences compared to a wild-type hepatitis virus X gene promoter and/or compared to a promoter of SEQ ID NO: 11, wherein said one or more differences results in restricted or prevention of transcription from said promoter. 
     
     
       17. The polynucleotide of  claim 5 , wherein:
 upon introduction into a eukaryotic cell, no polypeptide of a length greater than 12, 11, 10, 9, or 8 amino acids in length encoded by said variant X gene is produced; and/or 
 said polynucleotide is incapable of producing a polypeptide of a length greater than 12, 11, 10, 9, or 8 amino acids in length encoded by said variant X gene. 
 
     
     
       18. The polynucleotide of  claim 5 , wherein the variant X gene comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotide changes. 
     
     
       19. The polynucleotide of  claim 5 , wherein said modified PRE encodes an RNA that promotes nuclear RNA export and/or increases mRNA stability. 
     
     
       20. The polynucleotide of  claim 5 , further comprising a viral nucleic acid comprising a variant Flap, wherein the variant Flap contains a deletion of all or a portion of the nucleotides corresponding to the central polypurine tract (cPPT) and/or the central termination sequence (CTS) regions of a wild-type or unmodified Flap sequence. 
     
     
       21. The polynucleotide of  claim 20 , wherein the variant Flap comprises deletion of all or a contiguous portion of nucleotides corresponding to nucleotides in the cPPT region set forth in SEQ ID NO:123 and comprises deletion of all or a contiguous portion of nucleotides corresponding to nucleotides in the CTS region set forth in SEQ ID NO:124. 
     
     
       22. The polynucleotide of  claim 20 , comprising:
 a variant Flap comprising the sequence of SEQ ID NO: 122 or a sequence having at least at or about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:122; and 
 a modified PRE comprising the sequence of SEQ ID NO:29 or 126 or a sequence having at least at or about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:29 or 126. 
 
     
     
       23. The polynucleotide of  claim 5 , wherein the recombinant receptor is an antigen receptor and/or a chimeric receptor. 
     
     
       24. An expression cassette comprising the polynucleotide of  claim 5  and a promoter operably linked to the nucleic acid encoding the recombinant protein. 
     
     
       25. The polynucleotide of  claim 5 , wherein said stop codon is selected from among:
 a stop codon beginning at a nucleotide position corresponding to position 420 in the sequence set forth in SEQ ID NO:1; 
 a stop codon beginning at a nucleotide position corresponding to position 424 in the sequence set forth in SEQ ID NO:1; 
 a stop codon beginning at a nucleotide position corresponding to position 428 in the sequence set forth in SEQ ID NO:1; and 
 a stop codon beginning at a nucleotide position corresponding to position 432 in the sequence set forth in SEQ ID NO:1. 
 
     
     
       26. The polynucleotide of  claim 25 , wherein said stop codon is an amber (TAG) or opal (TGA) stop codon. 
     
     
       27. A vector comprising the polynucleotide of  claim 5 . 
     
     
       28. The vector of  claim 27 , which is a viral vector. 
     
     
       29. A cell comprising the polynucleotide of  claim 5 . 
     
     
       30. A virus particle, comprising the vector of  claim 27 . 
     
     
       31. A pharmaceutical composition comprising the cell of  claim 29  and a pharmaceutically effective carrier. 
     
     
       32. A method, comprising introducing the vector of  claim 27  to a cell, under conditions whereby expression of the recombinant protein is effected in the cell. 
     
     
       33. A polynucleotide, comprising a modified post-transcriptional regulatory element (PRE), said modified PRE comprising a variant of a wild-type hepatitis virus X gene, said variant X gene comprising a plurality of stop codons not present in the wild-type X gene, wherein said stop codon is selected from among:
 a stop codon beginning at a nucleotide position corresponding to position 420 in the sequence set forth in SEQ ID NO:1; 
 a stop codon beginning at a nucleotide position corresponding to position 424 in the sequence set forth in SEQ ID NO:1; 
 a stop codon beginning at a nucleotide position corresponding to position 428 in the sequence set forth in SEQ ID NO:1; and 
 a stop codon beginning at a nucleotide position corresponding to position 432 in the sequence set forth in SEQ ID NO:1. 
 
     
     
       34. The polynucleotide of  claim 33 , wherein said stop codon is an amber (TAG) or opal (TGA) stop codon. 
     
     
       35. The polynucleotide of  claim 33 , further comprising a nucleic acid encoding a recombinant protein operably linked to the modified PRE. 
     
     
       36. A vector comprising the polynucleotide of  claim 35 . 
     
     
       37. A cell comprising the polynucleotide of  claim 35 . 
     
     
       38. A pharmaceutical composition comprising the cell of  claim 37  and a pharmaceutically effective carrier. 
     
     
       39. A polynucleotide, comprising a modified post-transcriptional regulatory element (PRE), said modified PRE comprising a variant of a wild-type hepatitis virus X gene, said variant X gene comprising a sequence encoding a post-translational modification signal not present in the wild-type hepatitis virus X gene. 
     
     
       40. The polynucleotide of  claim 39 , further comprising a nucleic acid encoding a recombinant protein operably linked to the modified PRE. 
     
     
       41. A vector comprising the polynucleotide of  claim 40 . 
     
     
       42. A cell comprising the polynucleotide of  claim 40 . 
     
     
       43. A pharmaceutical composition comprising the cell of  claim 42  and a pharmaceutically effective carrier. 
     
     
       44. A polynucleotide, comprising a viral nucleic acid comprising a variant Flap, wherein the variant Flap contains a deletion of all or a portion of the nucleotides corresponding to the central polypurine tract (cPPT) and/or the central termination sequence (CTS) regions of a wild-type or unmodified Flap sequence. 
     
     
       45. The polynucleotide of  claim 44 , further comprising a nucleic acid encoding a recombinant protein operably linked to the variant Flap. 
     
     
       46. A cell comprising the polynucleotide of  claim 45 .